Department of Orthopaedic Surgery and Broad Stem Cell Center, University of California at Los Angeles, California, USA.
Department of Medicine, University of Indiana, Indianapolis, Indiana, USA.
Stem Cells. 2017 May;35(5):1273-1289. doi: 10.1002/stem.2599. Epub 2017 Mar 19.
Adipose tissue is a rich source of multipotent mesenchymal stem-like cells, located in the perivascular niche. Based on their surface markers, these have been assigned to two main categories: CD31 /CD45 /CD34 /CD146 cells (adventitial stromal/stem cells [ASCs]) and CD31 /CD45 /CD34 /CD146 cells (pericytes [PCs]). These populations display heterogeneity of unknown significance. We hypothesized that aldehyde dehydrogenase (ALDH) activity, a functional marker of primitivity, could help to better define ASC and PC subclasses. To this end, the stromal vascular fraction from a human lipoaspirate was simultaneously stained with fluorescent antibodies to CD31, CD45, CD34, and CD146 antigens and the ALDH substrate Aldefluor, then sorted by fluorescence-activated cell sorting. Individual ASCs (n = 67) and PCs (n = 73) selected from the extremities of the ALDH-staining spectrum were transcriptionally profiled by Fluidigm single-cell quantitative polymerase chain reaction for a predefined set (n = 429) of marker genes. To these single-cell data, we applied differential expression and principal component and clustering analysis, as well as an original gene coexpression network reconstruction algorithm. Despite the stochasticity at the single-cell level, covariation of gene expression analysis yielded multiple network connectivity parameters suggesting that these perivascular progenitor cell subclasses possess the following order of maturity: (a) ALDH ASC (most primitive); (b) ALDH ASC; (c) ALDH PC; (d) ALDH PC (least primitive). This order was independently supported by specific combinations of class-specific expressed genes and further confirmed by the analysis of associated signaling pathways. In conclusion, single-cell transcriptional analysis of four populations isolated from fat by surface markers and enzyme activity suggests a developmental hierarchy among perivascular mesenchymal stem cells supported by markers and coexpression networks. Stem Cells 2017;35:1273-1289.
脂肪组织是多能间充质干细胞样细胞的丰富来源，位于血管周围壁龛中。根据其表面标志物，这些细胞被分为两大类：CD31/CD45/CD34/CD146 细胞（外膜基质/干细胞 [ASCs]）和 CD31/CD45/CD34/CD146 细胞（周细胞 [PCs]）。这些群体表现出未知意义的异质性。我们假设醛脱氢酶（ALDH）活性，一种原始功能标志物，可以帮助更好地定义 ASC 和 PC 亚类。为此，从人体脂肪抽吸物的基质血管部分同时用荧光抗体染色 CD31、CD45、CD34 和 CD146 抗原和 ALDH 底物 Aldefluor，然后通过荧光激活细胞分选进行分选。从 ALDH 染色谱的末端选择的单个 ASC（n=67）和 PC（n=73）通过 Fluidigm 单细胞定量聚合酶链反应对一组预定的标记基因进行转录谱分析（n=429）。对这些单细胞数据，我们应用了差异表达和主成分和聚类分析，以及原始基因共表达网络重建算法。尽管在单细胞水平上存在随机性，但基因表达分析的协变产生了多个网络连接参数，表明这些血管周围祖细胞亚类具有以下成熟顺序：（a）ALDH ASC（最原始）；（b）ALDH ASC；（c）ALDH PC；（d）ALDH PC（最原始）。该顺序通过特定的类特异性表达基因组合独立支持，并通过相关信号通路的分析进一步证实。总之，通过表面标志物和酶活性从脂肪中分离的四种细胞群体的单细胞转录分析表明，血管周围间充质干细胞之间存在发育层次结构，由标志物和共表达网络支持。干细胞 2017；35:1273-1289。
